Cell culture products are used to grow large numbers of desired cells for medical testing, research and other uses. Certain materials, typically polymers, are optimal for use as substrates in cell culture products and are typically employed. For example, polystyrene is cheap, optically clear, and may be processed at low temperatures. Large molecular weight cell attachment proteins (CAP's) are bound to substrate surfaces. Cells are then grown on the CAP-coated surfaces. The cells adhere to the CAP's and the CAP's promote cell growth. The CAP's bind readily to substrate materials such as polystyrene. However, CAP's are very expensive and have short shelf lives, even at sub-freezing temperatures.
In the last few years, it has been shown that certain regions of these CAP's--small peptides--can mimic the cell adhesion and growth promoting action of the CAP's. The primary advantage of these peptides is their greater stability--they can be stored at room temperatures for long periods of time. However, unlike the CAP's, the unmodified peptides do not adhere well to substrate materials such as polystyrene. In order to obtain sufficient surface concentration of these cell attachment peptides (cap's) on substrate materials, the material's surface must be chemically modified. Most materials used as substrates for cell culture products are relatively stable and difficult to chemically modify. Typical methods of chemical modification include strong acids and toxic solvents or plasma or corona discharge treatments. Once the material is modified to accept cap's, it is said to be "activated".
Dialdehyde starch (DAS) is a commercially available compound made from starch by oxidation with periodic acid. DAS has been used in medicinal and bio-active compositions. U.S. Pat. No. 3,495,000 discloses sustained release medicinal compositions adapted for peroral administration using a matrix comprising an admixture of dialdehyde starch and polyvinylpyrrolidone, polyvinyl chloride or ethyl cellulose. The medicament, dialdehyde starch, and polymer are mixed and granulated.
At an oxidation degree of 100%, DAS contains two aldehyde groups per original glucose unit. These aldehyde groups readily react with molecules containing amine, imine, or hydrazide groups. U.S. Pat. No. 3,706,633 discloses water-insoluble, enzymatically active compositions prepared by condensing dialdehyde starch with an alkylene diamine to produce a polymeric product, reducing and diazotizing the product, so as to produce a polymeric polydiazonium salt, and then coupling the product with an active enzyme.
DAS has also been utilized in photographic mixtures. U.S. Pat. No. 3,284,204 discloses an adhesive solution for bonding the hydrophobic film base and the hydrophilic emulsion in photographic film. The adhesive solution comprises gelatin, an alkylene glycol derivative of polymannuronic acid, dialdehyde starch, water and a water-miscible solvent or mixture of solvents.
An approach to activating surfaces of certain cell culture substrate materials which uses DAS and which is an alternative to chemical modification of a substrate is disclosed in Y. Ikada, H. Iwata, T. Mita, and S. Nagaoka, "Grafting of Proteins onto Polymer Surfaces with Use of Oxidized Starch", J. Biomed. Mater. Res., 13, 607-22 (1979). In that approach, DAS was grafted to PVA hydrogel and EVA film. The PVA and EVA contain hydroxyl groups. The DAS molecules were grafted onto the polymers through acetalization between the aldehyde and the hydroxyl groups. Biologically active molecules were then attached to the bound DAS. Alpha-amylase grafted onto the DAS-coated EVA film showed a distinct enzymatic activity in hydrolysis of amylose and starch, but the activity was very low compared with that of ungrafted, soluble alpha-amylase.
Although the method of Ikada et al allows specific cell culture substrate materials to be activated so as to accept cap's without chemical modification of the substrate materials, the method is taught only for polymers containing hydroxyl groups--which will chemically react with DAS. These polymers, such as EVA and PVA, are not optimal for cell culture substrate materials from a cost and processing standpoint. Furthermore, optimal activated substrate surfaces bind biologically active molecules without significantly hampering their activity. Therefore, a need exists for a method of attaching DAS to other surfaces, notably polystyrene, without chemical modification of the surfaces and without significantly deleteriously affecting the biological activity of biologically active molecules which may be attached.